This invention relates to the field of agriculture and biological control of insect pests. In particular, the invention provides an apparatus and methods for low-cost mass production of insecticidal nematodes.
Heightened awareness of the dangers of chemical insecticides, combined with passage of the Federal Food Quality Protection Act restricting usage of these insecticides, has further increased the need for alternative insect control measures. Use of biological insecticides, in particular entomopathogenic nematodes, are consequently becoming an increasingly viable alternative to control insect pest populations.
Nematodes are simple, colorless, unsegmented roundworms that lack appendages. They may be free-living, predaceous or parasitic. The entomopathogenic nematodes of the genera Steinernema and Heterorhabditits are insect parasitic nematodes that possess an optimal balance of attributes to enable their use as biological control agents for insect pests.
Steinernema and Heterorhabditis have similar life cycles. The non-feeding infective juvenile seeks out insect hosts and penetrate into the insect body. There the juvenile releases a symbiotic bacterium, (Xeneorhabdus for steinernematids, Photorhadbdus for heterorhabditids), which multiplies rapidly and causes insect death. The nematodes feed upon the bacteria and liquefying insect, and mature into adults. The life cycle is completed in a few days, and hundreds of thousands of new infective juveniles emerge from the insect cadaver in search of fresh insect hosts.
Numerous benefits are derived by the use of nematodes as opposed to chemical insecticides or other biological methods. For example, nematodes are lethal to a greater number of important soil insect pests but are completely safe for plants and animals. Further, most chemical insecticides and biologicals require days or even weeks to kill insect pests, whereas nematodes usually kill most insect pests within 24-48 hours.
The two genera of entomopathogenic nematodes comprise nearly 30 species that are useful as biological control agents against a large number of insect pests, including artichoke plume moth, root weevils, cranberry girdler, sciarids, wood borers, fungus gnats, scarabs, mole crickets billbugs, armyworm, cutworm and webworm.
Unfortunately, for nematodes to represent a commercially viable alternative to chemical insecticides a self-contained, low cost system of production is preferable if not necessary. However, current methods of nematode production present tedious multi-step procedures involving sterilization techniques and expensive equipment, resulting in high production costs and contamination problems.
Moreover, though some of the above-described systems have been used on a small scale, a major difficulty arises when scale-up of nematode production is attempted. In vivo systems have proven difficult to scale-up. Temperature and humidity are the only wholly controllable process parameters. Spraying insect hosts with infective juveniles can accelerate the inoculation step, or the insects can be dipped into a nematode suspension. But harvesting emerging infective juveniles has remained an intractable bottleneck. In the laboratory, the White trap has been the traditional method of collecting entomopathogenic nematodes. In the White trap, infected insect cadavers are placed within a topless petri plate and the plate is placed in a tray of water. As the nematodes complete their development and emerge to seek new insect hosts, they exit the cadaver into the tray of water and are unable to escape. Nematodes are thus collected in the tray water. Scale-up of harvesting has consisted of simply providing larger White traps, usually by placing several dishes within a large tray. Because nematode lateral migration from the host cadaver to the water reservoir is required, increasing dish diameter beyond 150 mm quickly becomes counterproductive.
Carne and Reed (1964) described a harvest apparatus in which cadavers would be supported on nematode-permeable disks resting in the mouth of a series of large funnels. Water level in the funnels would be maintained at cadaver height by a common constant-level device. Emerging infective juveniles would pass through the disk and settle to the funnel bottom where they would be collected by opening the stopcock. The key innovation of the system was the use of perforated plates, which nematodes could migrate through to the water reservoir, reducing the need for significant lateral migration. There are no reports that the design was tested, and its design does not lend itself to successful scale-up, inasmuch as the funnel system would be prone to clogging before a significant number of nematodes could be collected.
Other methods for nematode production involve sterilized culture vessels, expensive and time consuming development and sterilization of media for nematode growth, and the introduction of nematodes into a bacterial suspension into the sterile environment. Accordingly, an urgent need exists for improved apparatus and methods for mass production, harvest and storage of insecticidal nematodes.
The present invention features an apparatus and method for in vivo production of parasitic nematodes. In accordance with the method of the invention, nematodes are cultured within a natural insect host. The apparatus comprises at least one harvesting area, a water dispensing system that promotes harvest of nematodes from the host organisms, and a water collection and concentration system for nematode collection and storage. In a preferred embodiment, the harvesting area comprises reusable stackable perforated trays preferably constructed from a non-toxic material such as plastic or aluminum. The perforated material allows passage of dispensed water from the water dispensing system while retaining the nematode hosts. The perforations are sized to retain the host organisms and facilitate the passage of harvested nematodes carried within the dispensed water.
Inoculation of the host organism is achieved by a simple dip or spray process where the host organisms are supported by a perforated tray and dipped or sprayed with a nematode suspension. During the initial phase of nematode infection, the nematodes release bacteria that are symbiotes of the nematodes, but that kill the host organism within 24 to 48 hours. The nematodes then reproduce within the host organism. During this phase, while the nematodes feed off the bacteria decomposing host tissue, the incubation chamber is maintained at a temperature and humidity suitable for maximum nematode growth and utilization of bacterial and host insect cadaver food sources. Within a determinable number of days of the initial inoculation, the host organism bursts open, releasing adult nematodes. At this time, the incubation area is sprayed or otherwise contacted with water or another aqueous medium, washing the nematodes away from the host organisms and allowing their collection and concentration in bulk form.
The water dispenser system includes at least one nozzle that is positioned at an angle effective for the removal of the incubated nematodes from the infected host organisms. Water is released from the nozzle as a fine mist or spray ensuring removal of the nematodes from the host organism and not removal of the host organism itself. The perforated trays allow passage of water through the system thereby flushing the nematodes towards a collection area. Effluent generated by the water dispensing system and carrying the nematodes passes through the supporting trays towards a collection area consisting of a flat solid surface with side walls angularly placed to direct the flow of liquid toward a nematode concentration system and storage vessel.
Other features and advantages of the present invention will be understood by reference to the drawings and detailed description that follow.